Until recently, the heart has been viewed as a terminally differentiated organ with no capacity for new cardiac myocyte (CM) formation. This view appears to be incorrect, in that we and others have been able to isolate cardiac-derived progenitor cells (CDPCs) from human myocardium. Extending these results, our recent studies indicate that cells expressing the stem cell surface marker c-kit can be isolated from human hearts immediately after explantation and subsequently induced to differentiate into CM via short-term co-culture with neonatal rat ventricular myocytes (RVMs). Though we typically find more c-kit+ cells usually in failing vs. nonfailing hearts, the need to replace these failing hearts via transplantation highlights the inadequacy of native cardiac repair mechanisms. Based on these findings, our broad working hypothesis is that increased c-kit+ CDPCs in failing human hearts include both lineage-negative c-kit+ and c-kit+/CD45(dim-moderate) cells that are each capable of new myocyte formation in vitro. In this context, the objective of this proposal is to quantify and characterize these distinct subpopulations of stem/progenitor cells within human hearts with an emphasis on elucidating their functional capacity for replication and CM differentiation. Our first aim is to identify what types of stem/progenitor cells are present in normal and failing human hearts. We will define distinct stem/progenitor subpopulations based on immunotyping of disaggregated myocardial cells with fluorescence microscopy and flow cytometry and perform complementary studies in tissue sections from the same hearts to define their distribution. Our second aim is to characterize replicative capacity of the selected CDPC subpopulations based on a combination of static assays (telomere length, telomerase activity and and p16INK4a expression) and functional assessment of proliferation rates. Our third aim is to characterize the cardiac myogenic potential of selected CDPC subpopulations derived from human hearts. These studies will define the rates and frequency of CM differentiation for sorted subpopulations under stardardized co-culture conditions, define whether cell contact is required for induction of CM differentiation by neonatal rat myocytes and identify secreted factors (chemokines or growth factors) that promote or augment rates of in vitro CM diffentiation in selected CDPC subpopulations. The clinical/therapeutic significance of this proposal is based on the premise that insights into the proliferative and cardiomyogenic potential of endogenous cardiac stem/progenitor cell subpopulations will promote progress towards therapeutic cardiac regeneration with or without cell therapy per se. PUBLIC HEALTH RELEVANCE: In recent successful experiments, we have isolated resident stem/progenitor cells from human hearts and induced their in vitro differentiation into contracting cardiac myocytes. Building on these findings, this research is focused on characterizing selected subpopulations of these stem/progenitor cells with an emphasis on elucidating their capacity for replication and differentiation into functioning cardiac myocytes. We contend that insights into the cardiomyogenic potential of endogenous cardiac stem/progenitor cells will promote progress towards therapeutic cardiac regeneration with or without cell therapy per se.